8. The cryoprotection procedure is generally carried out at cold
temperatures, even though in some cases there is no clear proof
of this having a beneficial effect. Apart from reduced toxicity of
Me 2 SO, a reduced diffusion rate at low temperatures may have
a positive effect.
9. When transferring/pipetting the (not immobilized) suspension into the cryovials, occasionally shake the culture jar to
make sure that cells remain suspended. Since plant cell cultures
are always more or less clumpy, use a pipette tip with widened
opening. Furthermore, pipetting should be performed quickly
to avoid settling of cells, which may block the pipette tip during
emptying.
10. For each specific cell line, the cooling program has to be
adjusted to its needs. In the older literature, cooling rates of
approximately 1
C/min have been reported. More recently,
cooling rates of 0.5–0.25
C/min have been shown to yield
better results for most cell lines [12]. This is in agreement with
our experiences. Hold temperatures range from À35 to
À40
C, for durations of 5–20 min.
11. During the thawing procedure, water may enter into the vials
and cause microbial contaminations. We therefore recommend
the use of a sterilized water bath and/or at least sterilized
water.
12. In most cases, regrowth on solid medium is better than
regrowth in liquid medium.
References
1. Tabata H (2004) Paclitaxel production by
plant cell culture technology. Adv Biochem
Eng Biotechnol 87:1–23
2. Hellwig S, Drossard J, Twyman RM, Fischer R
(2004) Plant cell cultures for the production of
recombinant proteins. Nat Biotechnol
11:1415–1422
3. Ziv M (2005) Simple bioreactors for mass
propagation of plants. In: Hvoslef-Eide AK,
Preil W (eds) Liquid culture systems for
in-vitro plant propagation. Springer, Berlin,
pp 79–93
4. Toshiyuki N, Seichiro H, Inze ´ D (2010)
Tobacco BY2 cells. In: Lo ¨rz H, Widholm J
(eds) Biotechnology in agriculture and forestry.
Springer, Berlin
5. Kim KW, Bamba T, Harada K, Fukusaki E,
Kobayashi A (2006) Time course of metabolic
profiling in Arabidopsis thaliana cell cultures
after salt stress treatment. J Exp Bot
58:415–424
6. Murata J, Matsumoto E, Morimoto K,
Koyama T, Satake H (2015) Generation of
triple-transgenic Forsythia cell cultures as a
platform for the efficient, stable, and sustainable production of Lignans. PLoS One 10:
e0144519
7. Yang Y, Pu X, Qu X, Chen F, Zhang G, Luo Y
(2017) Enhanced production of camptothecin
and
biological
preparation
of
N1-acetylkynuramine in Camptotheca acuminata cell suspension cultures. Appl Microbiol
Biotechnol 101:4053–4062
8. Withers L, King P (1980) A simple freezing
unit and routine cryopreservation method for
plant cell cultures. CryoLetters 1:213–220
9. Hao YJ, You CX, Deng XX (2002) Effects of
cryopreservation on developmental competency, cytological and molecular stability of citrus callus. CryoLetters 23:27–35
10. Van Eck JV, Keen P (2009) Continued expression of plant-made vaccines following long644
Heinz Martin Schumacher et al.
temperatures, even though in some cases there is no clear proof
of this having a beneficial effect. Apart from reduced toxicity of
Me 2 SO, a reduced diffusion rate at low temperatures may have
a positive effect.
9. When transferring/pipetting the (not immobilized) suspension into the cryovials, occasionally shake the culture jar to
make sure that cells remain suspended. Since plant cell cultures
are always more or less clumpy, use a pipette tip with widened
opening. Furthermore, pipetting should be performed quickly
to avoid settling of cells, which may block the pipette tip during
emptying.
10. For each specific cell line, the cooling program has to be
adjusted to its needs. In the older literature, cooling rates of
approximately 1
C/min have been reported. More recently,
cooling rates of 0.5–0.25
C/min have been shown to yield
better results for most cell lines [12]. This is in agreement with
our experiences. Hold temperatures range from À35 to
À40
C, for durations of 5–20 min.
11. During the thawing procedure, water may enter into the vials
and cause microbial contaminations. We therefore recommend
the use of a sterilized water bath and/or at least sterilized
water.
12. In most cases, regrowth on solid medium is better than
regrowth in liquid medium.
References
1. Tabata H (2004) Paclitaxel production by
plant cell culture technology. Adv Biochem
Eng Biotechnol 87:1–23
2. Hellwig S, Drossard J, Twyman RM, Fischer R
(2004) Plant cell cultures for the production of
recombinant proteins. Nat Biotechnol
11:1415–1422
3. Ziv M (2005) Simple bioreactors for mass
propagation of plants. In: Hvoslef-Eide AK,
Preil W (eds) Liquid culture systems for
in-vitro plant propagation. Springer, Berlin,
pp 79–93
4. Toshiyuki N, Seichiro H, Inze ´ D (2010)
Tobacco BY2 cells. In: Lo ¨rz H, Widholm J
(eds) Biotechnology in agriculture and forestry.
Springer, Berlin
5. Kim KW, Bamba T, Harada K, Fukusaki E,
Kobayashi A (2006) Time course of metabolic
profiling in Arabidopsis thaliana cell cultures
after salt stress treatment. J Exp Bot
58:415–424
6. Murata J, Matsumoto E, Morimoto K,
Koyama T, Satake H (2015) Generation of
triple-transgenic Forsythia cell cultures as a
platform for the efficient, stable, and sustainable production of Lignans. PLoS One 10:
e0144519
7. Yang Y, Pu X, Qu X, Chen F, Zhang G, Luo Y
(2017) Enhanced production of camptothecin
and
biological
preparation
of
N1-acetylkynuramine in Camptotheca acuminata cell suspension cultures. Appl Microbiol
Biotechnol 101:4053–4062
8. Withers L, King P (1980) A simple freezing
unit and routine cryopreservation method for
plant cell cultures. CryoLetters 1:213–220
9. Hao YJ, You CX, Deng XX (2002) Effects of
cryopreservation on developmental competency, cytological and molecular stability of citrus callus. CryoLetters 23:27–35
10. Van Eck JV, Keen P (2009) Continued expression of plant-made vaccines following long644
Heinz Martin Schumacher et al.